The present invention relates to a method for purification of mitomycin C from the culture of a mitomycin C-producing microorganism.
Mitomycin C is an anti-tumor antibiotic obtained by culturing a microorganism belonging to the species Streptomyces caespitosus and has been clinically applied for many years.
For purification of mitomycin C from the culture obtained by culturing the microorganism described above, there are known the activated carbon adsorption method which comprises adding an activated carbon to the culture filtrate obtained by removing cells to adsorb mitomycin C thereon and eluting mitomycin C with an organic solvent; or a purification method which comprises extracting mitomycin C contained in the culture filtrate with an organic solvent, and subjecting the resulting concentrate of mitomycin C to alumina chromatography or counter current distribution (Method A: Japanese Published Unexamined Patent Application No. 17897/60, U.S. Pat. No. 3,660,578).
As an improvement of the above method, there has been known a method which comprises adsorbing the culture filtrate onto a reverse phase adsorption resin; eluting mitomycin C with a solvent such as acetone, methanol, ethanol, etc.; concentrating the eluate to remove the solvent; saturating the concentrate with sodium chloride to dissolve the saturated concentrate in chloroform; applying the chloroform extract to alumina column chromatography; eluting the adsorption zone of mitomycin C with methanol; and concentrating the eluate; adding ether, petroleum ether, benzine or ligroin to the concentrate to obtain crystals of mitomycin C (Method B: Japanese Published Examined Patent Application No. 9094/61).
Method A described above encounters such problems that operations such as the elution after adsorption onto activated carbon, the extraction with an organic solvent, etc. are inefficient and thus a recovery yield of mitomycin C is low, etc.
Also in Method B, for example, Duolite S-30 (Duolite Co., Ltd.) which is specifically shown as the reverse phase adsorption resin has a poor adsorbability of mitomycin C so that large quantities of the resin and solvent should be used. Upon the extraction with chloroform, extraction efficiency is low so that a large quantity of chloroform is used and the extraction operation is repeated. Furthermore, the steps of eluting mitomycin C and isolating mitomycin C from the alumina adsorption zone subsequent to chromatography involve complicated operations and are also undesired in view of working environments. In addition, the purity of the resulting eluate is not so high. The crystals obtained from the eluate are not satisfactory in purity, unless they are recrystallized. Moreover, a time period required for the overall steps of purification is prolonged. Thus, the prior art methods encounter various problems.
Therefore, an improve method is always in demand for purification of mitomycin C in an industrial scale, in view of operability, productivity, etc.